Academic Editors: Helmut Cölfen and Mei Pan

Abstract: Several crystal forms of valacyclovir hydrochloride, including two anhydrous and three hydrates, were investigated in this study. At the same time, a new hemihydrate of valacyclovir hydrochloride was first discovered and its properties were characterized by PXRD, TGA, DSC, and Raman in this study. The hemihydrate shows a distinctive PXRD pattern and a melting point of 209 ◦ C with a water weight loss of 2.42% from the thermal analysis. The Raman spectra show a few distinctive peaks in the region of 1250–1400 cm−1 due to different crystal forms. The thermostability testing suggests it is a stable crystal form and remain the same for several months under high temperature and humidity. All these crystal forms show good dissolubility in the water at room temperature with excess 100 mg/mL.

1. Introduction Polymorphism of a drug compound usually results from the possibility of at least two differentspatial arrangements of the molecules in the crystal lattice, or in some cases, from variations inmolecular conformation, including crystalline and amorphous forms as well as solvate and hydrateforms [1–4]. Due to difference in the solid–state structure, polymorphs can be imparted with different chemicaland physical properties, including melting point, chemical reactivity, apparent solubility, dissolutionrate, and optical and mechanical properties of solid dosage forms [5–7], which may affect subsequentprocess and/or manufacture of drug products. Valacyclovir hydrochloride (VCV), [L-Valine, 2-[(2-amino-1, 6-dihydro-6-oxo-9H-purin-9-yl)methoxy] ethyl ester, monohydrochloride], as shown in Figure 1, is the L-valyl ester prodrug ofacyclovir and has demonstrated antiviral activity against herpes simplex virus types, 1 (HSV-1) and 2(HSV-2) and varicella-zoster virus (VZV) both in vitro and in vivo [8–13]. Although VCV has been usedas one of the most important antiviral drugs, study on its polymorphism and pseudopolymorphism isstill in negligible quantity. So far, two anhydrous and several hydrates crystalline forms of valacyclovirhydrochloride were reported in the US patent 6107302, US 6849736 B2 and US 0021444 as well as theirpreparation processes [14–16]. Based on the complexity and influence on the subsequent drug dosage,it is necessary to further investigate the polymorphism of VCV.

From the diffraction patterns, one can see that Form I, II, IV and V are consistent as previously Crystals 2017, 7, 140 3 of 9reported crystal forms. However, the PXRD pattern of the Form III—with characteristic diffractionpeaks at 2θ 3.5◦the From ◦ , 7.9◦ , 8.5◦ , 9.3◦ , 13◦ , 14.4◦ , 16.3◦ , 20.8◦ , 24.5◦ , and 27.2◦ —is completely distinctive , 6.9diffraction patterns, one can see that Form I, II, IV and V are consistent as previouslyfrom reported those ofcrystalthe other forms.four forms.theIn However, particular, PXRD pattern ofForm III exhibited the Form III—with several verydiffraction characteristic characteristic at 6.9at◦ ,2θpeakspeaks 7.9 ◦ , 13◦ , 20.8◦ and 23.5◦ , where no any peak can be detected in the patterns of other 3.5°, 6.9°, 7.9°, 8.5°, 9.3°, 13°, 14.4°, 16.3°, 20.8°, 24.5°, and 27.2°—is completely distinctive fromforms. those of the It indicates thatother Form fourIII forms. should In particular, be a new Form III exhibited polymorphic form several veryIn of VCV. characteristic peaks this order to confirmpoint,atthermal 6.9°, 7.9°,analysis 13°, 20.8°including and 23.5°, where DSC and no any TGA peak can be detected technology wereinused the patterns of other to further forms. analyze theIt new indicatespolymorphic form. that Form III should be a new polymorphic form of VCV. In order to confirm this point, thermal analysis including DSC and TGA technology were used to further analyze the new polymorphic2.2. Differential form. Calorimetry Scanning The DSC thermograms 2.2. Differential of the five crystal forms of VCV are presented in Figure 2. For Form I, II, Scanning Calorimetryand III—different crystal forms—as hydrates containing a certain of crystal water, there are significant The DSC thermograms of the five crystal forms of VCV are presented in Figure 2. For Form I, II,endothermic peaks on crystal their DSC scans because ◦C and III—different forms—as hydratesofcontaining water lossa in the temperature certain rangethere of crystal water, of 60–140 are(Figure 2a). DSC significant curves ofpeaks endothermic FormonI andtheir II DSCshowed two partially scans because of wateroverlapping endothermic loss in the temperature rangepeaks of at70 and 130 °C◦ C respectively, which means there areIItwo different types ofoverlapping crystal water in the crystal 60–140 (Figure 2a). DSC curves of Form I and showed two partially endothermicstructure, peakswhereas at 70 andonly one 130 °C endothermic respectively, which event means at about there are ◦ Cdifferent 141two is seen for Form types III. Inwater of crystal addition, in thethere crystal structure, whereas only one ◦ endothermic event at about 141 °C is seenare not any endothermic peaks in 60–140 C on the runs of Form IV and Form V, which indicates that for Form III. In addition,both there of them are not are any endothermic anhydrous peaks in 60–140 °C on the runs of Form IV and Form V, which indicates compounds. that both of them are anhydrous The melt/decomposition events compounds. of the five forms were shown in Figure 2b. For Form I, the melting The melt/decomposition events of the five forms were◦ shown in Figure 2b. For Form I, the meltingand decomposition simultaneously occurs at about 205 C because there is only a single exothermic and decomposition simultaneously occurs at about 205 °C because there is only a single exothermic peakpeak on onthetheDSCDSCcurve, curve, while Forms II, IV, and V have two significant melting and decomposition while Forms II, IV, and V have two significant melting and decomposition stages,stages,respectively. Melting point point respectively. Melting is an important is an important parameter parameter for a polymorphic for a polymorphic drug, which to drug, somewhich to some extent canextentbecanused betoused to identify identify different different crystal form.crystal form. temperatures The melting The meltingoftemperatures the four forms of theestimated were four forms and wereestimated listed and listed in Table in Table 2. Form 2. Form III has III has transformation a solid-solid a solid-solid transformation at about 197 °C, at about which 197 ◦itC, means is which probably means already turned into another crystalline form after the dehydration process.it is probably already turned into another crystalline form after the dehydration process.

2.3. Thermogravimetric Analysis

2.3. Thermogravimetric Analysis The water contents of all the forms were determined by TGA and the curves are illustrated in The water contents of all the forms were determined by TGA and the curves are illustrated inFigure 3. The three hydrates showed significant water release steps from Figure 3a, and Form I andFigure 3. The three hydrates showed significant water release steps from Figure 3a, and Form I andII were respectively calculated to be 6.93 wt% and 4.78 wt% in the temperature range of 50–140 °C,II were respectively calculated to be 6.93 wt% and 4.78 wt% in the temperature range of 50–140 ◦ C,but it is 2.42 wt% for Form III in a higher temperature range of 123–145 °C. The above results arebut it is 2.42 wt% for Form III in a higher temperature range of 123–145 ◦ C. The above results arerespectively consistent with water content theoretically calculated from sesquihydrate, monohydrate,respectively consistent with water content theoretically calculated from sesquihydrate, monohydrate,and hemihydrate. Whereas it is clear that Form IV and V had no any weight loss in the sameand hemihydrate. Whereas it is clear that Form IV and V had no any weight loss in the sametemperature zone from Figure 3b, this suggest they are anhydrous crystal forms.temperature zone from Figure 3b, this suggest they are anhydrous crystal forms.

100 200 300 100 200 300

Temperature(℃) Figure 3. TGA Figure 3. TGA curves curves of VCV polymorphs: of VCV polymorphs: (a) (a) Form Form I, I, Form Form II II and and Form Form III; III; (b) (b) Form Form IV IV and and Form Form V, V, the the insert insert is is the the profiles profiles of of the the derivative derivative curves. curves.

In addition, In addition,thetheprofiles profilesofofthe thederivative derivative curves curves insert insert in Figure in Figure 3a show 3a show there there are two are two weightweight lossloss stagesstages for Forma for Forma I and IIIand whileII Form while III Form has III onlyhas only one. one.results These Theseare results arecorresponding totally totally corresponding with thewith profilesDSC the DSCshown profiles shown2.inInFigure in Figure 2. Inwater fact, each fact, each lease water lease stage stage usually usually stands stands for for a specific a specific combinationcombinationmode between mode water between and VCV water and VCV molecules. Thismolecules. suggests This suggests that there that bonding are two there aremodes two bonding amongwater with VCV molecular in VCV hydrates for Form I and II but only one for Form III. FormFormmodes among water with VCV molecular in VCV hydrates for Form I and II but only one for DSCIII. Formand TGA DSC and results ofTGA Formresults III, theofsingle Formwater III, theloss single water process loss process actually actually confirms confirms that it is a purethat it is a hydratepure hydratecrystalline crystalline form with halfform with crystal half crystal water. water.

2.4. Raman Spectroscopy

As well well known, known,Raman Ramanspectroscopy spectroscopy is aisreliable a reliable technology technology to differentiate to differentiate crystalcrystal forms.forms. FromFrom the Ramanthe Raman spectra spectra of theof theVCV five five VCVcrystalcrystal formsforms in Figure in Figure 4, the4,most the most distinctive distinctive region region can can be be seenbetweenseen 12501250 between and 1400 cm cm and 1400 −1 − 1 . In this. Inregion, FormForm this region, I and IVand show threethree V show peaks, FormForm peaks, II andII IV andhave four IV havepeaks,four but Form peaks, III has but Form IIIonly two peaks. has only two peaks. From Table 3, Form I has two peaks at 1308.5 and 1351.3 cm−1 with a small peak at 1392.3 cm−1 ,whereas Form II has three peaks in the region at 1299.4, 1331.7, and 1352.3 cm−1 with a small peak at1387.3 cm−1 . The new crystal Form III shows two peaks at 1307.1 and 1353.3 cm−1 . The anhydrateForm IV has four peaks at 1298.4, 1331.7, 1352.8, and 1386.8 cm−1 , whereas another anhydrate form Vshows three peaks at 1306.1, 1348.8, and 1393.36 cm−1 . It is clear that different crystal forms exhibitdistinctive Raman spectra.Crystals 2017, 7, 140 5 of 9Crystals 2017, 7, 140 5 of 9

From Table Table 3. Summary

3, Form I has twoof Raman peaksPeak Positions at 1308.5 andfor the Five 1351.3 cmForms −1 withofaValacyclovir. small peak at 1392.3 cm−1,whereas Form II has three peaks in the region at 1299.4, 1331.7, and 1352.3 cm−1 with a small peak at Raman Shift (cm−1 ) Form I Form II Form III Form IV Form V1387.3 cm−1. The new crystal Three peaks at 409.4,FormFour IIIpeaks shows two peaks at 409.9, at 1307.1 Three peaks at 471.5, and 1353.3 Four peaks atcm409.4, . TheFive −1 anhydrate Form peaks at 409.4, 438.1, 400–600 472.6 and 501.8 470.4, 493.1 and 536.4 504.0 and 527.7 439.9, 490.3 and 538.1 470.9, 503.5 and 578.6IV has four peaks at 1298.4, 1331.7, 1352.8, and 1386.8 cm , whereas another anhydrate form V shows −1 Four peaks at 630.7, Five peaks at 630.2, 642.1, Five peaks at 629.6, 642.6, Five peaks at 634.5, 643.2, Five peaks at 626.9, 940.9,three peaks at 1306.1, 600–800 1348.8, 644.8, 754.3 and 777.3and 1393.36 cm785.2 687.9, 751.2 and −1. It is clear that different crystal forms exhibit distinctive 691.1, 752.7 and 782.5 685.7, 754.8 and 783.1 692.2, 751.7 and 780.4

Raman spectra. 1000–1200 Five peaks at 1023.0, Three peaks at 1019.4, Four peaks at 1021.9, Four peaks at 1010.1, Four peaks at 1019.9, 1067.8, 1097.1 and 1171.3 1065.3 and 1177.8 1067.8, 1096.6 and 1176.3 1064.2, 1089.4 and 1176.3 1067.3, 1091.5 and 1172.3 Three peaks at 1308.5, Four peaks at 1299.4, Two peaks at 1307.1 and Four peaks at 1298.4, Three peaks at 1306.1, 1200–1400 Table 1351.3 3. andSummary 1392.3 of1331.7, Raman 1352.3Peak Positions and 1387.3 1353.3 for the Five Forms of Valacyclovir. 1331.7, 1352.8 and 1386.8 1348.8 and 1393.3 Five peaks at 1416.7, Six peaks at 1411.3, Five peaks at 1420.7, Six peaks at 1457.4, Five peaks at 1416.2, 1400–1700 Raman 1450.0, 1479.7, 1571.0 1454.5, 1473.8, 1569.5, 1452.5, 1480.7, 1570.6 1473.8, 1565.1, 1607.0, 1451.0, 1477.7, 1568.5 andForm 1629.3 I Form 1606.6 and II 1666.5 and Form 1629.3 III 1633.6Form IV and 1667.0 Form V and 1625.9 Shift (cm−1) Three peaks atFour peaks at Three peaks at Four peaks at Five peaks at2.5. Apparent Solubility 400–600 409.4, 472.6 and 409.9, 470.4, 493.1 471.5, 504.0 and 409.4, 439.9, 490.3 409.4, 438.1, 470.9, 501.8 and 536.4 527.7 and 538.1 503.5 and 578.6 As well known, Four peakstheat solid-state structure Five peaks at has Fivea peaks significant at influence Five peaksonat the apparent Five peakssolubility atof600–800 the drug substance. 630.7, 644.8, In this study, the apparent 630.2, 642.1, 687.9, solubility 629.6, 642.6, 691.1, of polymorphic 634.5, 643.2, 685.7,solids of VCV were 626.9, 940.9, 692.2, 754.3 and 777.3 751.2 and 785.2 752.7 and 782.5 754.8 and 783.1measured and listed in Table 4. It is obvious that all the crystal forms have excellent dissolubility with 751.7 and 780.4 Five peaks atexcess 100 mg/mL in the ultrapure water. Three peaks at However, theattwo anhydrous Four peaks Four peaksForms—IV at Fourand V—show peaks at 1023.0, 1067.8, 1000–1200best apparent solubility 1019.4, 1065.3 and 1021.9, 1067.8, 1010.1, 1064.2, 1019.9, 1067.3, 1097.1 and with about 100 mg/mL compared with the other hydrates. The results of the 1177.8 1096.6 and 1176.3 1089.4 and 1176.3 1091.5 and 1172.3present study are consistent with 1171.3 the commonly observed trend that the aqueous solubility of hydratecompounds can be significantly Three peaks at lesspeaks Four thanat their anhydrous Two peaks at forms [17]. Four peaks at Three peaks at 1200–1400 1308.5, 1351.3 1299.4, 1331.7, 1298.4, 1331.7, 1306.1, 1348.8 and 1307.1 and 1353.3 and 1392.3 Table 4. Apparent Solubility of all the Forms of VCV in Water (25 C). 1393.3 1352.3 and 1387.3 1352.8 and 1386.8 ◦ Five peaks at Six peaks at Five peaks at Six peaks at Five peaks at 1416.7, 1450.0, 1411.3, 1454.5, 1420.7, 1452.5, 1457.4, 1473.8, 1416.2, 1451.0, 1400–1700 Crystals Apparent Solubility 1479.7, 1571.0 1473.8, 1569.5, (mg/mL) Crystal 1480.7, 1570.6 andform after 1607.0, 1565.1, Solubility Determination 1477.7, 1568.5 and Form Iand 1629.3 1606.6 145 and 1666.5 1629.3 1633.6 and 1667.0 Form I 1625.9 Form II 143 Form II Form Solubility2.5. Apparent III 118 Form III Form IV 147 Form I As well Formknown, V the solid-state 161 structure has a significant influence Form on theVapparent solubility ofthe drug substance. In this study, the apparent solubility of polymorphic solids of VCV weremeasured Once and listed in Table the solubility 4. It is obvious measurements hadthat beenallcompleted, the crystal forms have excellent the remaining dissolubility crystals with were collectedexcessand 100 mg/mL subjected to PXRDin the ultrapure testing. water. suggest The results However, the that two anhydrous Forms I, II, III, andForms—IV and V—show V did not transform intoother crystals forms, but Form IV spontaneously converted to Form I during the time resultsbest apparent solubility with about 100 mg/mL compared with the other hydrates. The of the of solubilitypresent studyItareequilibrium. consistent is easily with the commonly to understand observed why solubility trend of Form IVthat the aqueous is close to thosesolubility hydrates.of hydratecompounds can be significantly less than their anhydrous forms [17]. Form V 161 Form V

Once the solubility measurements had been completed, the remaining crystals were collectedand subjected to PXRD testing. The results suggest that Forms I, II, III, and V did not transform intoother crystals forms, but Form IV spontaneously converted to Form I during the time of solubilityCrystals 2017, 7, 140 6 of 9equilibrium. It is easily to understand why solubility of Form IV is close to those hydrates.

2.6. Stability2.6. Stability Studies Studies Stability of Stability of aa drug drug compound compound is is also also an an important important property property during during pharmaceutical pharmaceutical production. production.A stability study for Form III was performed by storing someA stability study for Form III was performed by storing some sample under sample under a condition of 40of°C, a condition 4075% ◦ C,RH for 16 weeks. During the testing, several batches of specimens were in turn75% RH for 16 weeks. During the testing, several batches of specimens were in turn taken out at taken out at intervalsfor PXRDforandintervals PXRDDSCand measurements DSC measurements to monitor their structure to monitor changes. their structure As shown changes. in Figure As shown 5a, 5a, in Figure noimpurity diffraction peaks were detected in the PXRD patterns of all the specimens,no impurity diffraction peaks were detected in the PXRD patterns of all the specimens, which means which means thecrystalthe structure crystal structurestillstill retained its original retained form. its original form.

From Figure 5b,

5b, broad broadendothermic endothermicpeaks peakscan canbebeseen inin seen the temperature the range temperature of 50–110 range ◦ C the °C on of 50–110 onDSCDSCthe curves of specimens curves after of specimens stored after forfor stored four fourweeks, weeks,which whichmeans meansaawater water lease lease process. process. However,considering relevant unchanged unchanged PXRD patterns, patterns, the presence of the water does not change change the the crystal crystalstructure of the hemihydrate, so it should be not crystal water but absorption water. Instructure of the hemihydrate, so it should be not crystal water but absorption water. In addition, addition, it isit is noticed that no recognizable distinction was viewed from the melting points of all the specimens,which further suggests that their crystal form is not changed after a long storage time. Above resultsindicate that hemihydrate of VCV has good stability and suitable for use in solid drug dosage.

with a purity exceeding 99.5%. All aqueous solutions were prepared using Milli-Q deionized (DI)Water with a resistivity of 10.2 MΩ·cm and total organic content <5 ppb.

3.2. Preparation of Polymorphs and Hydrates

The five crystal forms—including two anhydrous and three hydrates—were prepared and namedas Forms I, II, III, IV, and V respectively (as shown in Table 5).

Table 5. The Five Crystal Forms of VCV, Including Two Anhydrous and Three Hydrates.

Number Form I Form II Form III Form IV Form V

Crystal form Sesquihydrate Monohydrate Hemihydrate Anhydrous one Anhydrous two

Form I, II, IV, and V of VCV could be obtained by recrystallizing from the mixed solvent of waterand isopropanol. The VCV solution with 20 wt% raw VCV was first heated up to 80 ◦ C and refluxedfor one hour then cooled down to room temperature until crystals precipitation in full. During thisprocess, Forms I, II, IV, and V can be prepared respectively by adjusting the percentage of water inmixed solvent. In fact, Form I can be obtained from mixed solvent with 10 wt% water, Form II in awater content range of 6–8 wt%, and Form IV when the water content is lower than 5%. In particular,Form V was obtained in the mixture with 8% water content by quenching to zero. For the preparation of Form III, 1 g VCV raw material was dissolved in 10 mL ethanediol atroom temperature, the solution was then heated up to 40 ◦ C under stirring. A mixture of 20 mLtrichloromethane and 20 mL isopropanol was prepared and cooled to zero. The VCV solution was thendropped into the mixed solvent with a rate of 1 mL/min, the crystals would settle out soon during thisprocess. At last, the precipitation was filtered and dried in an oven at 50 ◦ C.

3.3. Powder X-ray Diffraction Measurements

Powder X-ray diffraction (PXRD) patterns of all the forms of VCV were collected on a powderX-ray diffractometer (Thermal ARL X’TRA, Thermal Technology Co., Santa Rosa, CA, USA) equippedwith Cu Kα radiation (λ = 1.54178 Å), operating with a 40 mA current and 40 kV voltage. The datawas recorded at a continuous scanning rate of 1.2◦ /min over a 2θ angular range of 3–40◦ with a 0.02◦step size.

3.4. Thermal Analysis

Thermal analysis was carried out using a differential scanning calorimetry (PYRIS DiamondDSC, PerkinElmer Co., Waltham, MA, USA) calibrated using the indium and zinc standards. TheseVCV samples were respectively weighted and encapsulated in aluminum pans and then heated from40–250 ◦ C at a heating rate of 5◦ /min under nitrogen gas flow of 20 mL/min. Thermogravimetric analysis (TGA) was performed on a thermogravimetric analyzer (PYRIS 1TGA, PerkinElmer Co., Waltham, MA, USA) which is calibrated using standard weight and the Curietemperature of magnetic metal. About 4 mg of samples were placed in a platinum crucible and heatedfrom ambient temperature to 300 ◦ C at a rate of 10 ◦ C/min with a 20 mL/min dry nitrogen purge.

3.5. Raman Measurement

Raman spectra were recorded on a */PI Action Tri Vista Raman microscope (Princeton Instruments,Trenton, NJ, USA) equipped with a 20× objective and utilizing a 633 nm laser. The scan range was 100to 3600 cm−1 , using three scans of 30 s length each per spectrum.

3.7. Stability Testing

In order to evaluate the stability of the new crystal form product, a long-term stability testingwas carried out under certain temperature and humility. During this process, Form III sample wasstored under an accelerated condition of 40 ◦ C and 75% RH for 16 weeks. The crystal transformationof the sample, including crystal structure and thermal behavior, was monitored periodically by PXRDand DSC.

4. Conclusions In this study, a few of VCV crystal forms were obtained by recrystallization in different mixedsolvents. At the same time, a new polymorphic form—hemihydrate of VCV—was first discovered andits physicochemical properties were investigated by PXRD, DSC, TGA, and Raman. The PXRD pattern for the new crystal form is completely different from the other polymorphsreported previously. It has melting point measured to be 209 ◦ C from the DSC curve, and a weight lossof 2.42 wt% calculated from TGA profile in the temperature range of 123–145 ◦ C, which confirms thatit is a hemihydrate of VCV. The stability testing suggested the new polymorphic form is a stable crystal form and will remainthe same for several months under high temperature and humidity. The results suggest that it haspotential to be an active pharmaceutical ingredient (API) using in the drug dosage.

Acknowledgments: The authors would like to express the gratitude to the financial support from the YoungResearchers Foundation of Zhejiang Provincial Top Key Academic Discipline of Chemical Engineering andTechnology, Zhejiang Sci-Tech University (ZYG2015007), Zhejiang Provincial Natural Science Foundation underGrant No. LY16B010002.Author Contributions: Guoqing Zhang, Shuai Zhang, and Mengqing Zhou conceived and designedthe experiments; Shuai Zhang, Mengqing Zhou, Meiqi Zheng, and Zhaoxia Zhang performed theexperiments; Shuai Zhang and Mengqing Zhou analyzed the data; Tian Chen and Naixing Wang contributedreagents/materials/analysis tools; Shuai Zhang, Mengqing Zhou, and Guoqing Zhang wrote the paper.Conflicts of Interest: The authors report no conflicts of interest and are responsible for the content and writing ofthe paper.